Reconstitution of Golgi disassembly by mitotic Xenopus egg extract in semi-intact MDCK cells.

Abstract

Semi-intact cells are cells with plasma membranes that have been permeabilized by bacterial pore-forming toxins or surfactants. The addition of mitotic Xenopus egg extract to semi-intact cells can reconstitute a number of intracellular events that occur specifically at the onset of mitosis. In this chapter, we describe methods for reconstituting the disassembly of the Golgi apparatus by introducing mitotic Xenopus egg extract into semi-intact Mardin-Darby canine kidney (MDCK) cells. The Golgi apparatus was visualized in the cells by expression of green fluorescence protein (GFP)-tagged galactosyltransferase, a marker of trans-Golgi cisternae. Xenopus egg extracts arrested at mitosis or interphase were then prepared and added to the semi-intact MDCK cells. Disassembly of the Golgi apparatus was induced by mitotic Xenopus egg extract. This system can be used not only to elucidate the factors that are involved in the reconstitution process, but also to dissect the process into several elementary steps morphologically and biochemically.